Lower Vmax Values Research Articles

Identification of essential amino acids in phenylalanine ammonia-lyase by site-directed mutagenesis.

The postulated precursor of the prosthetic dehydroalanine of phenylalanine ammonia-lyase (PAL), serine 202, was changed to cysteine by site-directed mutagenesis. After cloning and heterologous expression in Escherichia coli, the gene product was assayed for PAL activity. Mutant S202C showed full catalytic activity, and its kinetic constants and the amount of thiol groups were identical to those of wild-type PAL. It must be concluded that in a posttranslational modification both water and hydrogen sulfide can be eliminated from the amino acid in position 202 to form dehydroalanine. In an attempt to identify further amino acids essential either for the posttranslational modification or for catalysis, arginine 174, glutamine 425, and lysine 499 were changed to isoleucine. Analysis of the heterologously expressed mutated gene products revealed that only the R174I mutant showed a significantly lower Vmax value (1/450) identifying this arginine as important. This finding was supported by treatment of wild-type PAL and mutant R174I with phenylglyoxal and 2,3-butandione. Both react specifically with the guanidino group of arginine. They irreversibly inhibited wild-type PAL but had no influence of the Vmax value of mutant R174I. Preincubation with l-phenylalanine protected wild-type PAL from inhibition by phenylglyoxal indicating that arginine 174 is close to the active site. Incubation with KCN irreversibly abolished the remaining activity of mutant R174I leading to the conclusion that arginine 174 is important in catalysis.

Characterization of cellular accumulation and toxicity of illudin S in sensitive and nonsensitive tumor cells.

Illudins are novel low molecular weight natural products cytotoxic to human tumor cells in vitro. Illudin-derived analogs are effective against experimental human cancers nonresponsive to conventional anticancer agents. It is not known why some illudin analogs are more efficacious in vitro and in vivo than other analogs. Therefore, the in vitro cytotoxicity of the parent compound illudin S towards tumor cells was characterized using radiolabeled drug. Two cell lines sensitive at nanomolar concentrations using only a 15-min exposure period displayed a saturable, energy-dependent accumulation of illudins with relatively low K(m) and high Vmax values. A nonsensitive cell line, requiring millimolar concentrations to achieve in vitro toxicity, showed minimal illudin uptake with higher K(m) and lower Vmax values. No release of radioactivity could be demonstrated from tumor cells, indicating that there was no efflux of illudin S (or metabolites) from these cells. The number of intracellular illudin S molecules required to kill 50% of cells of different tumor cell lines varied from 78000 to 1114000 molecules per cell and was correlated with the 2-h IC50 value determined using a colony-forming assay. Illudin S was cytotoxic to a variety of multidrug-resistant tumor cell lines regardless of whether resistance was mediated by gp170/mdrl, gp180/MRP, GSHTR-pi, topoisomerase I, topoisomerase II, increased DNA repair capacity, or alterations in intracellular thiol content. Information obtained in this study could be used to design clinical phase I trials and to develop analogs with improved therapeutic indexes.

OMEPRAZOLE HYDROXYLATION BY CYP2C19 AND CYP3A4: PREDICTION TOWARDS HUMAN LIVER ACTIVITIES USING THE DATA OF RECOMBINANT P450 ENZYMES

Omeprazole 5-hydroxylation and sulfoxidation activities were determined in liver microsomes of different humans whose levels of individual forms of cytochrome P450 (P450 or CYP) varied. Omeprazole 5-hydroxylation by liver microsomes of a human sample which contained relatively high levels of CYP3A4 and low levels of CYP2C19 were inhibited very significantly by ketoconazole and anti-CYP3A4 antibodies, while a human sample having high in CYP2C19 and low in CYP3A4 was found to be sensitive towards fluvoxamine and anti-CYP2C9 antibodies. Both recombinant human CYP2C19 and CYP3A4 enzymes had activities for omeprazole 5-hydroxylation, with low Km and high Vmax values for the former enzyme and high Km and low Vmax values for the CYP3A4. Omeprazole 5-hydroxylation activities of different human samples were found to be related to predicted values calculated from the kinetic parameters of recombinant enzymes and the levels of liver microsomal P450 enzymes. When recombinant human CYP2C19 and CYP3A4 were mixed at levels found in different human samples, relatively similar profiles of omeprazole oxidation by the recombinant and microsomal enzyme systems were determined. These results suggest that both CYP2C19 and CYP3A4 are involved in the 5-oxidation of omeprazole (at a substrate concentration of 10 μM) in human liver microsomes and that contributions of these P450 enzymes depend on the compositions of CYP2C19 and CYP3A4 in liver. Different contributions of CYP2C19 and CYP3A4 In the oxidation of testosterone and progesterone by human liver microsomes are also reported.

The Common I172N Mutation Causes Conformational Change of Cytochrome P450c21 Revealed by Systematic Mutation, Kinetic, and Structural Studies

We have investigated the structure and function of P450c21 with regard to a conserved site around Ile-172 by site-directed mutagenesis making single amino acid substitutions of residues 169 173. Substitutions of Ile-171 and -172 resulted in production of mutant proteins with dramatic reductions in enzymatic activities, indicating the importance of these two residues in maintaining the structure and function of P450c21. The I171N protein was present at a slightly lower level, due to a decreased rate of protein synthesis. The I172N apoprotein was synthesized at the normal rate, but its heme-bound P450 form was present at a much lower level. This I172N protein was tightly integrated into the membrane of endoplasmic reticulum, similar to the wild type P450c21, as shown by immunofluorescence detection, alkaline extraction, and cellular fractionation. Kinetic studies indicated that I172N had a lower Vmax value. In addition, the I172N protein was more sensitive to proteinase K digestion, indicating a possible alteration of conformation. This conformational change may result in the lower yield of the I172N hemoprotein and the reduced catalytic activity.

Synthesis of novel Se-substituted selenocysteine derivatives as potential kidney selective prodrugs of biologically active selenol compounds: evaluation of kinetics of beta-elimination reactions in rat renal cytosol.

Eighteen Se-substituted selenocysteine derivatives were synthesized as potential kidney selective prodrugs which can be activated by renal cysteine conjugate beta-lyase to selenium-containing chemoprotectants or antitumor agents. Selenocysteine derivatives with aliphatic and benzylic Se-substituents were synthesized by reducing selenocystine to selenocysteine followed by a reaction with the corresponding alkyl and benzyl halogenides. Selenocysteine derivatives with aromatic Se-substitutes were synthesized by reaction of beta-chloroalanine with substituted phenylselenol compounds, which were formed by reducing substituted diphenyl diselenides by NaBH4. The enzyme kinetic parameters (apparent Km and Vmax) of the beta-elimination reaction of the selenocysteine conjugates were studied in rat renal cytosol. The results suggest that Se-substituted L-selenocysteine conjugates are extremely good substrates for renal cysteine conjugate beta-lyases as indicated by low apparent Km and high Vmax values. The benzyl-substituted Se-conjugates appeared to be better substrates than the phenyl- and alkyl-substituted Se-conjugates. Corresponding L-cysteine S-conjugates were too poor substrates to obtain proper enzyme kinetics. Recently, local activation of cysteine S-conjugates by renal cysteine conjugate beta-lyases was proposed as a new strategy to target antitumor agents to the kidney. The present results show that Se-substituted selenocysteine conjugates may be more promising prodrugs because these compounds are much better substrates for beta-lyase.

Catalytic roles of the distal site asparagine-histidine couple in peroxidases.

There are highly conserved hydrogen bonds between the distal His and the adjacent Asn in many peroxidases. Although the crystal structure of horseradish peroxidase C (HRP) is not available, comparison of the amino acid sequence with cytochrome c peroxidase indicates that Asn70 is making the hydrogen bond with the distal His in the active site of HRP. To investigate the catalytic roles of the hydrogen bond, Asn70 in HRP was replaced with Val (N70V) or Asp (N70D). Though UV-vis, CD, and 1H-NMR spectra of native (plant enzyme), wild-type (recombinant enzyme), and mutant HRPs suggest that the active site and secondary structure are very similar even after mutation, the mutants exhibit low Vmax values for the hydroquinone oxidation (native, 281; wild-type, 283; and N70V, 18; and N70D, 33 microM.min-1). The rates of compound I formation were decreased to less than 10% of that of the native enzyme. The reduction rates of compounds I and II by guaiacol also were reduced to less than 10% of that of the native enzyme. Substituent effects of various phenol derivatives on the reduction of native, wild-type, and mutant compound I were examined. Large negative Hammett rho values (rho N70V:fast = -4.0, rho N70V:slow = -3.6, rho N70D = -3.8, rho native = -6.9, and rho wild-type = -6.8) are an indication of electron transfer being the rate-determining step in the phenol oxidation. However, these results also indicate the participation of the deprotonation step in the compound I reduction process. The proton abstraction from phenol must be harder for the mutants due to the decrease of basicity of the distal His upon mutation. Contrary to phenol oxidation, ABTS [2,2'-azinobis(3-ethybenzothiazoline-6-sulfonic acid)] oxidation activity was substantially increased by the mutations (native, 73; wild-type, 71; N70V, 217; and N70D, 234 microM.s-1). The redox potentials of N70V and N70D compounds II are 957 and 970 mV (vs NHE), which are 95 and 108 mV higher than that of native compound II (862 mV), respectively. Therefore, the high ABTS oxidation activities of mutants are attributed to these high redox potentials of compound II.

Protein Kinase C: Structure, Function, and Regulation

The protein kinase C family of enzymes transduces the myriad of signals promoting lipid hydrolysis. The prevalence of this enzyme family in signaling is exemplified by the diverse transduction mechanisms that result in the generation of protein kinase C's activator, diacylglycerol. Signals that stimulate members of the large families of G protein-coupled receptors, tyrosine kinase receptors, or non-receptor tyrosine kinases can cause diacylglycerol production, either rapidly by activation of specific phospholipase Cs or more slowly by activation of phospholipase D to yield phosphatidic acid and then diacylglycerol(1, 2, 3) . In addition, fatty acid generation by phospholipase A2 activation modulates protein kinase C activity(3) . Thus, multiple receptor pathways feeding into multiple lipid pathways have the common end result of activating protein kinase C by production of its second messenger.

A study of the substrate specificity of aminopeptidase N from Lactococcus lactis subsp. cremoris Wg2.

A systematic study was made of the ability of aminopeptidase N from Lactococcus lactis subsp. cremoris Wg2 to hydrolyse different peptide substrates. The enzyme showed a marked preference for substrates containing arginine as the N-terminal residue but, to a lesser extent, was also capable of cleaving other residues such as lysine and leucine. There was a tendency for the activity to increase with the hydrophobicity index of the C-terminal residue of dipeptide substrates. It was also observed that the enzyme tended to have higher affinities but lower Vmax values for tripeptides with hydrophobic C-terminal residues. The values determined for Km and Vmax increased with chain length for oligopeptides of the general formula Lys-Phe-(Gly)n, the optimum, as determined from Vmax/Km, being when n = 4. Typical Km values for the most effective substrates were in the range 0.2-0.6 mM.

Kinetic Properties of Adenine Nucleotide Analogues Against Purified 5-Phosphoribosyl-1-pyrophosphate Synthetases fromE. coli, Rat Liver and Human Erythrocytes

Abstract The nucleoside analogue 2′,3′-dideoxyadenosine (ddA), the phosphonate isostere of 2′,3′-dideoxy-2′,3′-didehydro-adenosine (d4A) 5′-monophosphate (d4API), and the acyclic nucleoside phosphonates PMEoA, PMEA, FPMPA and PMPA are potent and selective antiretroviral agents. We found that these compounds are recognized as substrates by the PRPP synthetases from E. coli, rat liver and human erythrocytes, as their monophosphate and triphosphate form in the reverse and forward reaction, respectively. In particular, ddA-5′-monophosphate (ddAMP) and ddA-5′-triphosphate proved to be excellent substrates for the enzymes. D4API was a relatively good substrate of the rat liver and human erythrocyte PRPP synthetases. The acyclic nucleoside phosphonates were rather poor substrates, as evident from their low Vmax values. None of the PRPP synthetases are found to act stereospecifically: they recognized both the S- and R-enantiomers of FPMPA and PMPA in a comparably efficient manner. Our data indicate that PRPP synt...

Structural connectivity in actin: effect of C-terminal modifications on the properties of actin

In this study, we use fluorescent probes and proteolytic digestions to demonstrate structural coupling between distant regions of actin. We show that modifications of Cys-374 in the C-terminus of actin slow the rate of nucleotide exchange in the nucleotide cleft. Conformational coupling between the C-terminus and the DNasal loop in subdomain II is observed in proteolytic digestion experiments in which a new C-terminal cleavage site is exposed upon DNasel binding. The functional consequences of C-terminal modification are evident from S-1 ATPase activity and the in vitro motility experiments with modified actins. Pyrene actin, labeled at Cys-374, activates S-1 ATPase activity only half as well as control actin. This reduction is attributed to a lower Vmax value because the affinity of pyrene actin to S-1 is not significantly altered. The in vitro sliding velocity of pyrene actin is also decreased. However, IAEDANS labeling of actin (also at Cys-374) enhances the Vmax of acto-S-1 ATPase activity and the in vitro sliding velocity by approximately 25%. These results are discussed in terms of conformational coupling between distant regions in actin and the functional implications of the interactions of actin-binding proteins with the C-terminus of actin.

Structural requirements for phosphorylation of myosin regulatory light chain from smooth muscle.

Site-directed and chimeric mutations of myosin regulatory light chains were used to identify residues important for phosphorylation of Ser19 by smooth muscle myosin light chain kinase. Arg16 and hydrophobic residues C-terminal of Ser19 in smooth muscle light chain were important substrate determinants in the intact protein. However, changes in the kinetic properties with mutations in the light chain were substantially smaller than results reported with structurally similar synthetic peptide substrates. These results together with the low Vmax value for short peptide substrates containing the consensus phosphorylation sequence suggest that there may be additional sites of interactions between the kinase and protein substrate. Chimeras of skeletal and smooth muscle light chains were constructed with exchanges at the N terminus and subdomains I, II, III, and IV. Analysis of results obtained on the kinetic properties for phosphorylation showed that subdomains I and II contribute to high Vmax values. Thus, a region distant from the consensus phosphorylation sequence in smooth muscle light chain is also an important substrate determinant for myosin light chain kinase.

Efficient autophosphorylation and phosphorylation of the beta-subunit by casein kinase-2 require the integrity of an acidic cluster 50 residues downstream from the phosphoacceptor site.

Various beta-mutants were investigated either as subunits or as substrates for casein kinase 2 (CK-2), in the absence of presence of polylysine. A total of 21 beta-mutants were characterized for their susceptibility to autophosphorylation, by combining them in equimolar amounts with the recombinant alpha-subunit. Six mutants, i.e. beta A5,6, beta A59-61,63,64, beta A55,57, beta 55-57, beta delta 171-215, and beta delta 150-215 exhibited a > 70% reduction in autophosphorylation. This strongly suggests that in addition to amino acid residues 5,6, distant amino acid residues within the sequence 55-64 are also involved in the process of autophosphorylation, possibly by means of a loop formation. The results obtained with the COOH-terminal-deleted mutants support the view that reconstitution of a functional holoenzyme must occur to allow efficient autophosphorylation. Polylysine prevents the autophosphorylation of beta wt (86% inhibition) inducing a parallel increase of the alpha-subunit autophosphorylation. The autophosphorylation of all mutants, with the exception of beta A55-57 and beta A59-61,63,64, is also inhibited by polylysine (>64%). The alpha-subunit autophosphorylation is increased with all mutants reconstituting a tetrameric holoenzyme. Only with the three largest COOH-terminal deletion mutants beta delta 150-215, beta delta 171-215, and beta delta 181-215 is no significant alpha-subunit autophosphorylation observed. The phosphorylation of the beta-subunit mutants added in large molar excess to CK-2 holoenzyme (either native or recombinant) is also severely impaired by Ala for Glu/Asp substitutions at position 5,6 and in the 55-64 region and by the deletion of the COOH-terminal segments 150-215 and 171-215. Such a phosphorylation is inhibited by polylysine, with the exception of mutants beta delta 171-215 and beta delta 150-215, whose phosphorylation is conversely stimulated by polylysine. The decreased phosphorylation efficiency of those mutants that are poor substrates is invariably accounted for by lower Vmax values, whereas the affinity for CK-2 is actually increased (Km values lower than that of beta wt).

CHARACTERIZATION AND INHIBITION OF POLYPHENOL OXIDASE FROM PEARS (PYRUS COMMUNIS L. CV. BOSC AND RED)

Red pears had higher PPO activity, total phenolics and chlorogenic acid concentration than Bosc pears. PPO activity, and phenolics both decreased in fruits held at room temperature. pH and temperature optima for Bosc and Red pears PPOs were 5.0 and 5.5, and 20 and 23 C, respectively. 4-Methylcatechol, catechol and dopamine were good substrates for PPO from both pear cultivars; however, no activity was observed with any of the mono-hydroxy substrates studied. Higher Km and lower Vmax values were observed for Bosc pear PPO. Heating at 75 C for 30 min completely inactivated the enzyme from both cultivars. Heating at 55 and 65 C for the same duration resulted in partial inactivation (45-60%) of this enzyme. Ascorbic acid, L-cysteine, sodium metabisulfite and thiourea effectively inhibited browning due to pear PPOs.

Asp304 of Escherichia coli acid phosphatase is involved in leaving group protonation.

Site-directed mutagenesis was used to explore the role of potential proton donors in the mechanism of the Escherichia coli acid phosphatase that is encoded by the appA gene. Asp304 appeared to be the only carboxylic acid residue that is conserved in the protein sequences of the high molecular weight acid phosphatases. The mutations Asp304Ala and Asp304Glu were introduced into appA and the corresponding proteins were overexpressed in E. coli and purified to homogeneity. Only small decreases were observed for the Km values of the substrates p-nitrophenyl phosphate, fructose 1,6-diphosphate, and tripolyphosphate. However, Vmax was greatly decreased, and the magnitude of effect depended markedly on substrate. Both mutant proteins exhibited significantly lower Vmax values with fructose 1,6-diphosphate, which possesses a much poorer leaving group than do the other two substrates. The importance of the leaving group was further tested by using a number of phenyl and alkyl phosphate derivatives as substrates. A linear correlation was observed between log Vmax and the pKa of the substrate leaving group for catalysis by the Asp304Ala mutant enzyme. These results are consistent with partition experiments using ethylene glycol as an alternate nucleophile, which indicated that for the Asp304Ala protein, the formation of a phosphoenzyme intermediate is the rate-determining step, in contrast to the situation for the wild type enzyme and the His303Ala mutant. In the latter case, the rate-limiting step of the reaction is interpreted to be the breakdown of phosphoenzyme. It is concluded that Asp304, rather than His303, is involved in protonation of the substrate leaving group.

Phi 29 DNA polymerase active site. The conserved amino acid motif “Kx3NSxYG” is involved in template-primer binding and dNTP selection

phi 29 DNA polymerase shares with other alpha-like DNA polymerases several regions of amino acid similarity. Among them, the conserved region characterized by the amino acid motif "Kx3NSxYG" has been proposed to form part of the polymerization active site of alpha-like DNA polymerases. Mutants in phi 29 DNA polymerase residue Tyr390 of this conserved motif had been previously described to be affected in DNA-dependent dNTP binding. In this paper, the functional significance of this conserved motif is further studied by the analysis of mutants in conserved residues Asn387, Ser388, and Gly391. Residue Phe393 of phi 29 DNA polymerase has also been selected as target for site-directed mutagenesis because of its conservation within the group of alpha-like DNA polymerases from genomes that replicate by a protein-priming mechanism. Mutant N387Y was shown to be affected both in initiation and polymerization reactions, showing 3-fold higher Km value for dATP and more than 11-fold lower Vmax value than the wild-type enzyme in the initiation reaction; moreover, it was affected in enzyme-DNA translocation. Mutant S388G retained initiation and polymerization activities; interestingly, this mutation significantly increased the efficiency of dNTP incorporation in non-templated reactions. Mutation Gly391 to Asp abolished template-primer binding as shown by gel retardation assays; this mutant was drastically affected in template-dependent dNTP incorporation both in initiation and polymerization reactions, but the efficiency of the non-templated phi 29 terminal protein-deoxynucleotidylation was higher than with the wild-type protein. Mutation Phe393 to Tyr severely decreased initial binding to template-primer DNA molecules, resulting in a reduced activity in DNA primer-dependent polymerization reactions but not in phi 29 terminal protein-dependent ones.

The effects of cysteine mutations on the catalytic activities of the reverse transcriptase of human immunodeficiency virus type-1.

The reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) has only 2 cysteine residues at positions 38 and 280. In order to investigate the role of these cysteines in the structure and function of the enzyme, we have previously modified each of the cysteines to serines employing site-directed mutagenesis. Two of the mutant forms of HIV-1 RT, the single mutant of cysteine 280 and a double mutant with both cysteines modified, were purified. In the present study we have compared the catalytic properties of the DNA-polymerizing and the ribonuclease H (RNase H) functions of the two mutant RTs to those of the native enzyme. The results indicate that the single mutant RT closely resembles the wild type enzyme in almost all the catalytic functions tested. The double cysteine mutant RT, on the other hand, exhibits several unique features. First, the specific activities of the RNA- and DNA-directed DNA synthesis are significantly lower than the corresponding activities of the other two enzymes. This probably results from the lower Vmax values exhibited by the double mutant RT, since the Km values calculated for all enzymes were similar. Second, the most outstanding differences are associated with the RNase H activity of the double mutant RT. The specific activity of RNase H is about 4-fold higher than the wild type and the single mutant RTs. Furthermore, the heat stability of the RNase H function of the double mutated RT is at least 15-fold higher than that of the other two RTs. The substantial resistance to heat denaturation is apparent only for the RNase H activity, since the DNA polymerizing function of the double mutant RT is as sensitive to heat denaturation as the other two proteins.

Liver mevalonate 5-pyrophosphate decarboxylase is responsible for reduced serum cholesterol in stroke-prone spontaneously hypertensive rat.

Spontaneously hypertensive rat (stroke-prone) (SHRSP) has an interestingly low serum cholesterol level due to a reduced biosynthesis of cholesterol in the liver (Iritani, N., Fukuda, E., Nara, Y., and Yamori, Y. (1977) Atherosclerosis 28, 217-222). In this study, we examined the mechanism underlying the reduction of hepatic cholesterol biosynthesis in the rat. Our initial findings in SHRSP, as compared with normotensive Wistar Kyoto rat (WKY), showed that 1) the incorporation of [14C]acetate into cholesterol in the liver slices was markedly less, 2) 3-hydroxyl-3-methylglutaryl (HMG) CoA reductase activity was not reduced, and 3) the incorporation of [3H]mevalonic acid into both cholesterol and squalene was significantly less. The above initial findings suggested that the reduction in the hepatic cholesterol biosynthesis took place in one or more enzymatic processes starting with mevalonic acid and continuing to squalene. When the incorporation of [3H]mevalonic acid into phosphomevalonate derivatives was studied using an ion exchange column, only the radioactivity incorporated into isopentenyl-pyrophosphate (isopentenyl-PP) was less in SHRSP. Furthermore, the specific activity of diphosphomevalonate (mevalonate-PP) decarboxylase in the liver-soluble fractions was reduced 50% in SHRSP as compared with WKY. Kinetic studies using liver crude extracts indicated a lower Vmax value in SHRSP (SHRSP, 0.47; WKY, 2.05 nmol/min/mg), and an unchanged Km value (SHRSP, 18.2; WKY, 19.6 microM). The activity of mevalonate-PP decarboxylase was also found to be reduced in other tissues, including the brain, testis, small intestine, and cultured vascular smooth muscle cells. From the above observations, we concluded that the lower activity of mevalonate-PP decarboxylase was responsible for the reduced cholesterol biosynthesis in the liver of SHRSP.

Expression and Functional Study of Wild-Type and Mutant Human Cytochrome P450c21 inSaccharomyces cerevisiae

The most common cause of congenital adrenal hyperplasia is deficiency of cytochrome P450c21 (21-hydroxylase), which catalyzes the synthesis of adrenal steroids. We have cloned the human P450c21 cDNA into yeast expression vectors under the control of either the glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) promoter or the aldehyde-dehydrogenase (ADH) promoter. P450c21 RNA, protein, and enzyme activity can be detected, indicating that both promoters drive the synthesis of P450c21. The expressed P450c21 catalyzes the conversion of both of its substrates, with Km and Vmax values of 0.33 microM and 280 nmoles/hr.nmole of P450c21 protein for progesterone, and 0.23 microM and 450 nmoles/hr.nmole for 17-hydroxyprogesterone. These kinetic properties are similar to those of human P450c21 expressed in COS-1 cells. The microsomal fraction containing P450c21 exhibited an absorption peak at 450 nm upon binding to CO, demonstrating its hemoprotein nature. The CO-difference spectra indicated that there were about 0.08 nmole P450c21 hemoprotein/mg microsomal protein. Coupling this expression system with site-directed mutagenesis, the Asn-172 mutant of P450c21 had about 20-100 lower Vmax values; yet it retained normal affinity toward both substrates. This mutant protein also exhibited an altered absorbance with a peak at 420 nm rather than at 450 nm.

Relationship between seasonal patterns of platelet serotonin uptake and 3H-imipramine binding in depressed patients and normal controls

Chicz-DeMet, Aleksandra, Christopher Reist, and Edward M. DeMet: Relationship Between Seasonal Patterns of Platelet Serotonin Uptake and 3H-Imipramine Binding in Depressed Patients and Normal Controls. Prog. Neuro-Psychopharmacol. & Biol. Psychiat. 1991, 15 (1): 25–39. 1. 1. Significant seasonal variations were found in the velocity of serotonin (Vmax) uptake and the density of 3H-imipramine binding sites (Bmax) in blood platelets from normal controls. 2. 2. Peak 3H-imipramine ( 3H-IMI) binding was found in February whereas peak serotonin (5HT) uptake was found in June and these measures were not correlated in paired comparisons. 3. 3. Both Vmax and Bmax values of depressed patients deviated from the normal seasonal pattern with lower uptake and binding in the patient group. 4. 4. A comparison of Vmax and Bmax deviations from the normal patterns of uptake and binding revealed a significant correlation between these measures such that patients with low Vmax values were the same as those with low Bmax values. 5. 5. The results support previous claims that the 3H-IMI binding site may be closely associated with, or identical to, a 5HT transport carrier. 6. 6. A significant correlation between uptake and binding further suggests that a common defect may be responsible for observed decreases in Vmax and Bmax values of depressed patients.

The role of Leu-190 in the function and stability of adenylate kinase.

To elucidate the role of C-terminal region of chicken adenylate kinase (a single polypeptide consisting of 193 amino acid residues) in the catalysis and stability of the enzyme, a series of mutant proteins truncated in the C-terminal region has been prepared by successive replacements of the sense codons by a termination codon via site-directed mutagenesis. Removal of the three C-terminal residues did not affect the apparent Michaelis constants (Km values) for AMP and ATP, although the Vmax values decreased gradually in parallel with the length of the polypeptide chain. A sudden increase in Km values for substrates, in particular for ATP, was observed on removal of one additional residue (Leu-190), the Vmax value also being less than one-half of that of the mutant enzyme with 3 residues shorter than the wild-type enzyme. These results suggest the importance of the highly conservative Leu-190. Therefore, we further prepared the mutant enzymes through replacement of Leu-190 by a variety of other amino acid residues. They all had substantially lower Vmax values and decreased thermostabilities. Their apparent Km values for ATP also changed, whereas those for AMP were affected to a lesser extent. The hydrophobicity of amino acid residues at position 190 was found to positively correlate with the specificity constants (kcat/Km values) for ATP and also with the thermostability of the enzyme. The fluorescence emission of the Trp-190 mutant enzyme was quenched by the addition of ATP. It is suggested that the C-terminal residues, particularly those around Leu-190, are present in a hydrophobic region which may be involved in binding of ATP.